Kaposi's Sarcoma-Associated Herpesvirus ORF6 Gene Is Essential in Viral Lytic Replication

Can Peng,Jungang Chen,Wei Tang,Xulin Chen,Chunlan Liu

doi:10.1371/journal.pone.0099542

Can Peng, Jungang Chen + Show 3 more

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https://doi.org/10.1371/journal.pone.0099542

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Abstract

Kaposi's sarcoma associated herpesvirus (KSHV) is associated with Kaposis's sarcoma (KS), primary effusion lymphoma and multicentric Castleman's disease. KSHV encodes at least 8 open reading frames (ORFs) that play important roles in its lytic DNA replication. Among which, ORF6 of KSHV encodes an ssDNA binding protein that has been proved to participate in origin-dependent DNA replication in transient assays. To define further the function of ORF6 in the virus life cycle, we constructed a recombinant virus genome with a large deletion within the ORF6 locus by using a bacterial artificial chromosome (BAC) system. Stable 293T cells carrying the BAC36 (wild type) and BACΔ6 genomes were generated. When monolayers of 293T-BAC36 and 293T-BACΔ6 cells were induced with 12-O-tetradecanoylphorbol-13-acetate (TPA) and sodium butyrate, infectious virus was detected from the 293T-BAC36 cell supernatants only and not from the 293T- BACΔ6 cell supernatants. DNA synthesis was defective in 293T-BACΔ6 cells. Expression of ORF6 in trans in BACΔ6-containing cells was able to rescue both defects. Our results provide genetic evidence that ORF6 is essential for KSHV lytic replication. The stable 293T cells carrying the BAC36 and BACΔ6 genomes could be used as tools to investigate the detailed functions of ORF6 in the lytic replication of KSHV.

Highlights

Kaposi’s sarcoma associated herpesvirus (KSHV), known as human herpesvirus 8 (HHV-8), is a member of the gamma-2 herpesvirus family [1]
We genetically analyzed the importance of KSHV ORF6 for KSHV lytic growth by construction of an ORF6-null genome
It has been reported that CDV, an inhibitor of KSHV DNA replication, was used to dissect KSHV lytic gene expression into two components: genes expressed without DNA replication and those required it

Summary

Introduction

Kaposi’s sarcoma associated herpesvirus (KSHV), known as human herpesvirus 8 (HHV-8), is a member of the gamma-2 herpesvirus family [1] It is associated with Kaposi’s sarcoma (KS), primary effusion lymphoma and multicentric Castleman’s disease [2,3]. The virus expresses most or all of its genes and viral DNA is amplified via a rolling circle mechanism by utilizing its own DNA polymerase and other factors [7]. Increasing evidence suggests that the small percentage of viral lytic replication plays great roles in viral pathogenicity. This spontaneous reactivation directly contributes to viral tumorigenesis through generation of virions for further spread infection and production of homologs of cellular cytokines which act in a paracrine manner for tumor progression [8]

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